The field of the present invention pertains to a dry solid medium and method for collection of genetic material in a form suitable for storage and/or subsequent analysis. Specifically, the present invention provides for a sampling device and method for collecting genetic material through the use of the dry solid medium. The invention further provides for analysis of stored genetic material using methods that are suited for automated analyzing systems.
Blood containing genetic material to be analyzed has typically been transported from the place of removal from a human or animal to the place of analysis as purified genetic material, liquid whole blood, frozen whole blood or whole blood dried onto paper. All of the methods have disadvantages. Transport of genetic material in blood as dried, purified genetic material is most desirable, but it requires a high standard of technical assistance to be available at the place of removal from the human or animal. When technical assistance is not available at the place of removal, whole blood or other unpurified samples are usually sent to a central facility where the genetic material is purified.
Transport of liquid whole blood often involves the need for sterility of collection. Under some circumstances, this is extremely inconvenient, for example, where the sample is a heel-prick taken from an infant. The transport of liquid whole blood or frozen blood also demands temperature control and an appropriate transport system other than the regular postal system. This is true even before considering concerns about hygiene. In addition, problems with pathogens associated with whole blood, such as the HIV virus, generally rule out the transport of any potentially infectious liquid or frozen sample except under proper and expensive supervision.
Blood dried on filter paper is a proven alternative to the above procedures and it has been shown that genetic material can be extracted and isolated form dried whole blood spots in a form and in sufficient quantities for use in DNA analysis. McCabe, E. R. B., et al., xe2x80x9cDNA Microextraction From Blood Spots on Filter Paper Blotters: Potential Screening Applications to Newborn Screening,xe2x80x9d Hum. Genet. 75:213-216 (1987). But, this procedure still suffers from a number of disadvantages. For example, typically, there has been no deliberate and rapid destruction of blood associated pathogens. This creates a potential hazard for blood handling personnel. In addition, usually, there has not been deliberate inhibition of the processes that degrade the genetic material other than occurring by desiccation. However, slow desiccation, or a small degree of re-hydration under conditions of relatively high humidity, will allow the growth of DNA or RNA destroying microflora. Moreover, even in the presence of a bacteriostatic agent of the type that does not denature proteins, there are conditions that permit enzymatic-autolytic breakdown of the genetic material and some nonenzymatic breakdown of the genetic material. (Enzymatic-autolytic breakdown refers to the process whereby dying or damaged tissues, of either human, animal or parasite cells, activate enzymes that degrade their own components). Furthermore, there is typically considerable difficulty desorbing very high molecular weight DNA or RNA from paper, if this is required. Surface adsorption effects can cause losses of genetic material that may cause the preferential loss of the least degraded, i.e. the most desired class of DNA or RNA molecules.
Thus, there is a need for a safe, convenient and minimally labor intensive means for storage of a genetic material to be analyzed that is contained in a liquid sample.
However, even if a sample of genetic material is collected in a safe, convenient and reliable form for storage and subsequent analysis, there are also logistic problems, which arise when there are many different types of analysis to be performed on a collected sample. For example, polymerase chain reaction (PCR) analysis requires a different primer-pair for each specific analysis to be performed. Obviously, the problems tend to further increase when multiple samples are submitted for analysis.
Present methods for in-situ-processing based on the use of oligonucleotide primers, for example, PCR, rely on the stored genetic material being heated and cooled in reaction mixtures that have primers added to them at the time of beginning temperature cycling. In many types of analysis of genetic material, it is the primers that determine the particular specificity of a reaction. Because there is usually a primer pair for each conceivable type of analysis, there are an extremely large number of possible primers (for example, all the sequences within the genes of humans, animals and all other living organisms including the pathogens of humans and animals). Thus, in any centralized facility that receives multiple samples for analysis of genetic material using, for example, oligonucleotide primers, the logistic problems can be immense.
Automation of analysis of genetic material allows for increased numbers of samples to be processed more efficiently. However, automation of the analysis of genetic material still requires the automated system to have completely separate delivery devices for each different set of primers. Otherwise, the cross-contamination that may occur will be impossible to control.
Thus, in general, if reactions like PCR are to be carried out at a centralized location using automated systems and using presently known methods, the range of different sequences which can be analyzed at one time will be restricted by the physical problems of keeping a clean delivery system of pipettes, etc., or by the molecular problems inherent in using mixtures of diverse primers. Clearly, circumvention of this restriction would be advantageous for analyzing multiple samples of genetic material for different genetic sequences.
Finally, samples can be taken from various surfaces and areas to test samples for particular substances such as antigens or genetic material. Currently, common methods can involve utilizing a swab or swab-like device. Generally, once the sample is obtained the sample is transferred to another medium to perform any subsequent analysis. In most cases, the swab is capable of being used on any surface, including, but not limited to skin, wounds, flesh, body orifices such as the mouth, and any other living or nonliving surface. Most swabs used and known to those of skill in the art have the basic components of a narrow rod support in combination with a collecting medium positioned and attached adjacent to the support.
The present invention provides a safe, convenient and minimally labor intensive apparatus and method for storage of a genetic material that is contained in a liquid medium. The present invention specifically provides for a sampling device and method for collecting genetic material through the use of a dry solid medium. The invention further provides for storage of genetic material in such a way as to allow for simplified analysis of one or more samples of genetic material using automated systems.
The invention provides a dry solid medium for storage of a sample of genetic material. The dry solid medium of the invention is composed of a solid matrix and a composition which when applied to the dry solid medium protects against degradation of genetic material stored on the dry solid medium. The dry solid medium further provides for inactivation of microorganisms, including those that may be pathogenic to humans.
The composition of the dry solid medium includes a weak base, a chelating agent, and anionic detergent and optionally, uric acid or a urate salt.
According to the invention, genetic material stored on the dry solid medium may be analyzed using methods known in the art, for example, polymerized chain reaction (PCR), ligase chain reaction (LCR), reverse transcriptase initiated PCR, DNA or RNA hybridization techniques including restriction fragment length polymorphism (RFLP) and other techniques using genetic or DNA or RNA probes, genomic sequencing, enzymic assays, affinity labeling, methods of detection using labels or antibodies and other similar methods.
Genetic material stored on the dry solid medium of the invention may be analyzed in situ or after removal from the dry solid medium.
Another embodiment of the invention provides a dry solid medium for storage of a sample of genetic material and includes a component that is functional in the subsequent analysis to be performed on the stored sample of genetic material. According to the invention, components for subsequent analysis which may be included with a sample of genetic material include, for example, nucleotide sequences such as PCR primers, target sequence stabilizers, genetic probes, primers for genetic sequencing or sets of oligonucleotide substrates for LCR analysis.
The invention also provides methods for using the dry solid media of the invention. In one embodiment, the invention provides a method for storing a sample of genetic material on the dry solid for storing a sample of genetic material on the dry solid medium in a substantially non-degraded form.
The invention further provides a method for storing a sample of genetic material and subsequently analyzing the stored sample of genetic material. According to this embodiment of the invention, the stored genetic material is preferably washed to remove any proteins or hemoglobin associated with a sample of genetic material or to remove any component of the composition of the dry solid medium which may inhibit subsequent analysis of the genetic material. The invention provides for the stored genetic material to be washed using either an aqueous or a non-aqueous washing system.
The aqueous and non-aqueous washing systems used to wash a sample of genetic material prior to analyzing the genetic material provide for removing protein, hemoglobin or components of the composition of the dry solid medium without substantially affecting the stored genetic material or any component included on the dry solid medium for subsequent analysis of the genetic material. In addition, the aqueous and non-aqueous wash systems of the invention may be used to wash genetic material that is stored, for example, on a solid matrix which does not have sorbed to it the DNA protecting composition of the invention.
The non-aqueous wash system of the invention includes contacting a sample of genetic material stored on the dry solid medium of the invention with a herein-described single-phase phenol solution. The single-phase phenol solution is then removed from the dry solid medium containing a sample of genetic material and the dry solid medium containing a sample of genetic material is then contacted with an aqueous alcohol wash solution. The aqueous alcohol wash solution is removed and the dry solid medium containing the sample of genetic material may further be washed with an aqueous ionic solution. The washed sample of genetic material may then be analyzed by methods recited above.
A sample of genetic material stored on a dry solid media of the invention may also be analyzed using standard enzymic tests used, for example, to detect phenylketonuria (PKU) or galactosemia after neutralizing the effects of protein denaturing components of the dry solid medium using, for example, a converter solution.
The dry solid medium and the dry solid medium including components for subsequent analysis is particularly useful for analysis of a genetic sample using automated systems.
A further embodiment of the present invention is the use of the dry solid medium as a swab device used to obtain and gather genetic material samples. Additionally, the present invention provides for a method of collecting genetic material samples utilizing the swab device.